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Student Number 90224018
Author Wan-Ching Won(翁婉青)
Author's Email Address morganww@yahoo.com.tw
Statistics This thesis had been viewed 2574 times. Download 2260 times.
Department Life Science
Year 2002
Semester 2
Degree Master
Type of Document Master's Thesis
Language zh-TW.Big5 Chinese
Title Proteomic Profiling of Arsenite-arrested Metaphase Cells
Date of Defense 2003-06-18
Page Count 57
Keyword
  • arsenite
  • metaphase Cells
  • Abstract Abstract
    Arsenic is a well-documented human carcinogen, primarily based on the evidence of clinical observation and epidemiological studies. Previous studies in our laboratory have shown that treatment of HeLa S3 cells with 5 μM arsenite leads to approximately 35% of the cells arrested in the mitotic metaphase. Treatment of arsenite-arrested mitotic metaphase cells with staurosporine (SSP, a protein kinase inhibitor) enforces them exiting from mitosis. In this thesis, experiments were conducted to ask how SSP enforces the mitotic exit. The microscopic structure of mitotic spindles and chromosomes were examined by immunofluorescent technique using anti-β-tubulin antibody and DAPI, and the content of Pds1 were analyzed by Western blot technique. The results show that treatment of arsenite-arrested mitotic cells with 50 nM SSP leads to degradation of Pds1 and increases in the frequency of cytokinesis. The proteins modified by SSP on arsenite arrested mitotic cells are analyzed by two-dimensional electrophoresis. These proteins are identified by MALDI-TOF technology. Several proteins are apparently modified by staurosporine treatment, for instance, carbamoyl-phosphate synthase, vinculin, chaperone, intermediate filaments related protein etc. Two-dimensional electrophoresis also detects differentially expressed proteins between the nocodazole and arsenite-arrested mitotic spindle fractions. These proteins are ubiquinol-cytochrome C reductase complex core protein I, intermediate filaments and chaperone related protein. The involvement of these proteins in mitotic arrest warrants further investigation.
    Table of Content 目 錄
                                    頁
    目錄…………………………………………………………………………….…..I
    『表』目錄……………………………………………………………………….IV
    『圖』目錄………………………………………………………………………..V
    中文摘要………………………………………………………………………....VI
    英文摘要…..…………………………………………………………………….VII
    第一章 緒論…..…………………………………………………………………..1
     壹、砷化物的簡介…..………………………………………………….………2
       一、砷的分佈與來源………………………………………………………2
       二、砷化物對人類健康之影響……………………………………………2
     貳、砷化物的細胞毒性與遺傳毒理…………………………………………..3
       一、砷化物的細胞毒性……………………………………………………3
       二、砷化物的遺傳毒理……………………………………………………3
     參、砷化物對細胞週期之影響…………………………………………………3
       一、細胞週期………………………………………………………………3
       二、砷化物對細胞週期之影響……………………………………………5 
       三、微管動力學……………………………………………………………5
       四、砷化物對有絲分裂期之影響…………………………………………6 
     肆、Staurosporine的介紹………………………………………………………7
    一、Staurosporine的簡介…………………………………………….….7
    二、Staurosporine的作用……………………………………………..…7
     伍、Pds1的介紹  ……………………………………………………………8
     陸、研究動機與實驗目的………………………………………………………8
    第二章 材料與方法……………………………………………………………..10
    壹、細胞培養…………………………………………………………………10
    1.細胞株…………………………………………………..……………10
    2.細胞培養………………………………………………….………….10
    貳、細胞分裂中期指數的檢測及計算…………………………..…………..10
    1.藥物處理及染色體製備……………………………………………..10
    2.細胞分裂中期指數的計算…………………………………………..11
    參、紡錘體的免疫螢光染色  …………………………………………….11
    肆、西方點墨法分析Pds1含量..……………………………………………11
    1.樣品製備………………………………………………..……………11
    2.蛋白質定量………………………………………………….……….12
    3.西方點墨法…………………………………………………………..12
    伍、二維電泳法………………..…………………………………………….12
    1. 全細胞樣品製備…………………………………..…………………12
    2. 紡錘體樣品製備……………………………………..………………13
    3.蛋白質裝填……………………………………..……………………13
    4.第一維等電交集電泳程式設定……………………..………………14
    5.平衡………………………………………………….……………….14
    6.第二維SDS-PAGE電泳…………………………………………….14
    陸、膠體染色法及影像處理…………………………………………………15
    1.SYPRO Ruby染色法………………………………………………..15
    2.影像處理…………………………………………………………….15
    柒、膠體內分解胜肽片段……………………………………………………15
    1.挖取膠點…………………………………………………………….15
    2.膠點褪染…………………………………………………………….15
    3.膠體內分解胜肽片段……………………………………………….16
    捌、蛋白質資料庫比對………………………………………………………16
    玖、影像分析…………………………………………………………………17
    第三章 結果……………………………………………………………………..18
    壹、亞砷酸鈉對紡錘體型態及染色體分佈的影響………………………….18
     貳、Staurosporine 對於亞砷酸鈉誘引之分裂中期停滯細胞離開有絲分裂期的影響……………………………………………………………………19
     參、Staurosporine對於亞砷酸鈉誘引之分裂中期停滯細胞之紡錘體的影響…………………………………………………………………………19
     肆、亞砷酸鈉誘引之分裂中期停滯細胞經staurosporine處理後之Pds1蛋白質的表現情形……………………………………………………………20
     伍、以二維電泳法觀察亞砷酸鈉誘引之分裂中期停滯細胞經staurosporine處理後之蛋白質修飾作用………………………………………………20
     陸、以二維電泳法觀察nocodazole以及亞砷酸鈉誘引之分裂中期停滯細胞內蛋白質的改變…………………………………………………………21
    第四章 討論……………………………………………………………………..23
    第五章 結論與展望……………………………………………………………..29
     壹、結論………………………………………………………………………29
     貳、展望………………………………………………………………………29
    參考文獻………………………………………………………….……………...44
    附錄……………………………………………………………………………....54

    『表』目錄

    表1. 亞砷酸鈉誘引之分裂中期停滯細胞經staurosporine不同時間處理後細胞
    內蛋白質的變化.………………………………………………………...31
    表2. 亞砷酸鈉及nocodazole誘引之分裂中期停滯細胞內蛋白質的變化…………………………………………………………………………32

    『圖』目錄
                                  頁
    圖1. 以免疫螢光染色法觀察亞砷酸鈉干擾紡錘體型態及染色體的分布
    …………………………………………………………………….……..33
    圖2. SSP促使亞砷酸鈉誘引之分裂中期停滯細胞離開有絲分裂期
    …………………………..……………………………………………….34圖3. 以免疫螢光染色法觀察SSP促使亞砷酸鈉誘引之分裂中期停滯細胞進 
    行細胞質分裂…………………………………………………………...35
    圖4. SSP促使亞砷酸鈉誘引之分裂中期停滯細胞進行細胞質分裂………………………………..……………………………………….36
    圖5. 亞砷酸鈉誘引之分裂中期停滯細胞經SSP處理後Pds1蛋白之表現受抑制…………………………………………………….…………………...37
    圖6. SSP處理亞砷酸鈉誘引之分裂中期停滯細胞的二維電泳膠片…..…….38
    圖7. 亞砷酸鈉誘引之分裂中期停滯細胞經SSP處理後,表現量增加的蛋白質:vinculin及glycyl-tRNA synthetase………………………………..39
    圖8. 亞砷酸鈉誘引之分裂中期停滯細胞經SSP處理後,表現量減少的蛋白質:carbamoyl-phosphate synthase……………..……….………………40
    圖9. 亞砷酸鈉誘引之分裂中期停滯細胞經SSP處理後,可能進行去磷酸化作用之蛋白質:75 kDa glucose regulated protein及heat shock 70 kDa protein 1…………………………………………………………………..41
    圖10. 細胞經亞砷酸鈉及nocodazole處理後的二維電泳膠片
    ……………………………………………………………………………42
    圖11. 細胞經亞砷酸鈉(5 µM, 24 h)及nocodazole (0.2 µM, 4 h)個別處理後,細胞間絲相關蛋白質:cytokeratin 8及vimentin的profile(側面圖)具差異性……………………………………………………………………...….43
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  • Te-Chang Lee(李德章)
  • Rong-Nan Huang(黃榮南)
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    Date of Submission 2003-07-09

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